Attaining the correct conformation and structure is essential for the proper function of proteins. In the cell, protein folding into its native structure is assisted by enzymes that accelerate folding and by chaperones that inhibit aggregation. Protein misfolding contributes to Alzheimer's disease, Parkinson's disease, prion-mediated infection, emphysema, and cystic fibrosis. Misfolding also limits the effective therapeutic use of recombinant proteins. Our long-term goal is to understand and mimic the biochemistry and enzymology of folding assistants. This proposal focuses on the in vitro and in vivo enzymology of protein disulfide isomerase (PDI), a folding catalyst and chaperone of the endoplasmic reticulum (ER) that is essential for the correct formation and isomerization of protein disulfides. The following specific aims are proposed. Specific Aim 1. PDI specificity and its multi-domain structure. The multidomain structure of PDI is essential for catalysis of isomerization, but substrate interactions that contribute to PDI recognition and catalysis have not been identified. Data from arrays of substrate peptides suggest a model in which PDI interactions are focused on positively charged substrate sites that pose barriers to isomerization and that these sites are located on multiple PDI domains. The model will be tested by mutagenesis of lysine and arginine residues in well-defined PDI substrates and the binding of domain deletion mutants of PDI to peptide arrays of substrate sequences. Specific Aim 2. Isomerization and reductive pathways in the yeast ER. The current model for ER disulfide formation suggests that PDI forms and breaks substrate disulfides and that oxidized and reduced PDI are both needed for proper folding. Recently discovered isomerase-deficient PDI mutants that still support wild-type yeast growth will be used to test models for maintaining ER redox balance. Experiments will also test for compensation pathways to supplement PDI activity, including the induction of non-essential PDI homologues in the ER and the more general unfolded protein response. Specific Aim 3. Mechanisms of isomerization. The factors that make PDI an effective catalyst of isomerization are not understood. To determine if PDI provides a faster pathway of isomerization involving reduction and reoxidation, PDI will be presented with specific folding intermediates of bovine pancreatic trypsin inhibitor (BPTI) which undergo slow, intramolecular isomerization in the absence of PDI. PDI mutants and single turnover experiments with wild-type PDI will detect if PDI provides faster isomerization by changing the mechanism. [unreadable] Mutations that alter the thiol/disulfide exchange kinetics and thermodynamics of the PDI active site will be used to determine which features of the chemistry of the active site contribute to catalysis. [unreadable] [unreadable]